Abstract

Neuronal L-type calcium channels are essential for regulating activity-dependent gene expression, but they are thought to open too slowly to contribute to action potential-dependent calcium entry. A complication of studying native L-type channels is that they represent a minor fraction of the whole-cell calcium current in most neurons. Dihydropyridine antagonists are therefore widely used to establish the contribution of L-type channels to various neuronal processes and to study their underlying biophysical properties. The effectiveness of these antagonists on L-type channels, however, varies with stimulus and channel subtype. Here, we study recombinant neuronal L-type calcium channels, CaV1.2 and CaV1.3. We show that these channels open with fast kinetics and carry substantial calcium entry in response to individual action potential waveforms, contrary to most studies of native L-type currents. Neuronal CaV1.3 L-type channels were as efficient as CaV2.2 N-type channels at supporting calcium entry during action potential-like stimuli. We conclude that the apparent slow activation of native L-type currents and their lack of contribution to single action potentials reflect the state-dependent nature of the dihydropyridine antagonists used to study them, not the underlying properties of L-type channels.

Here, we study recombinant neuronal CaV1.2 and CaV1.3 channels and show that these L-type calcium channels open over a range of voltages and activate rapidly in response to a variety of stimuli including individual action potential waveforms. Our data suggest that certain properties attributed to neuronal L-type channels, such as slow activation, are more reflective of the state-dependent action of the dihydropyridine antagonists used to study them and not the kinetics of native L-type currents.

L-type channels open rapidly

CaV1.3 L-type channels opened and closed with fast kinetics relative to CaV1.2 channels. Examples of superimposed, normalized representative currents for each channel demonstrate that activation rates of CaV1.3, CaV2.2, and CaV3.1 currents are comparable and faster compared with CaV1.2 (Fig. 1b,c). Activation time courses of CaV1.3 currents were indistinguishable from CaV2.2 over a range of test potentials, whereas CaV1.2 currents opened with significantly slower kinetics (Fig. 1c). The voltage dependence of CaV3.1 activation kinetics was steeper compared with CaV1.3 and CaV2.2 (Fig. 1c). Although slower compared with the other calcium channels, CaV1.2 channels opened with a time course more rapid than is typically reported for native dihydropyridine-sensitive L-type currents (Mermelstein et al., 2000). CaV1.2, CaV1.3, and CaV2.2 channels all closed rapidly and significantly faster than CaV3.1 T-type channels. T-type channels displayed characteristic slow deactivation tails (Fig. 1b, supplemental Fig. 1, available at www.jneurosci.org as supplemental material).

L-type channels are activated by action potential stimuli

The rapid gating of CaV1.2 and CaV1.3 channels suggested that these channels should respond well to brief action potential-like stimuli. We confirmed this using action potential waveforms as command voltages to evoke currents from cells expressing CaV1.2, CaV1.3, and CaV2.2 channels (holding potential, -80 mV) (Fig. 2a) (McCobb and Beam, 1991). Action potential waveforms, derived from sympathetic neurons, induced CaV1.3 currents with time courses indistinguishable from CaV2.2, whereas CaV1.2 channels opened with a slightly longer delay (Fig. 2a). The same result was obtained using an action potential waveform from a Purkinje neuron as the command voltage (supplemental Fig. 1a, available at www.jneurosci.org as supplemental material). The average time delay between the peak of the command voltage waveform and the peak of the inward calcium current was close to 0.6 ms for both CaV1.3 and CaV2.2 channels, compared with close to 0.8 ms for CaV1.2 (Fig. 2a,c). Action potential-evoked currents carried by CaV3.1 channels peaked more slowly (0.85 ms), similar to CaV1.2, but most notably, these channels deactivate slowly and permit calcium influx for several milliseconds after the membrane potential returns to -80 mV (McCobb and Beam, 1991) (supplemental Fig. 1b,c, available at www.jneurosci.org as supplemental material).

To compare the efficiency among CaV1.2, CaV1.3, and CaV2.2 channels to support calcium entry, we calculated total charge moved in response to single action potential waveforms. Total charge was expressed relative to peak current amplitude evoked by step depolarizations for each cell. This normalized for differences in expression efficiencies among the clones (Fig. 2b). CaV1.3 channels were at least as efficient as CaV2.2 channels in supporting calcium entry during action potential-like stimulation, and CaV1.2 channels were slightly less efficient (Fig. 2d). The use of peak current amplitudes to normalize for differences in expression efficiencies slightly underestimates CaV1.3 values relative to CaV2.2. This is because CaV1.3 channels open at voltages negative to CaV2.2, where the driving force on calcium is greater.

Our results clearly show that single action potentials can activate both CaV1.2 and CaV1.3 L-type calcium channels. Given this, why are single action potentials apparently so inefficient at recruiting L-type calcium channels (Bonci et al., 1998; Mermelstein et al., 2000; Brosenitsch and Katz, 2001; Yasuda et al., 2003)? We considered the possibility that the use of dihydropyridines in studies of native neuronal L-type channels has greatly underestimated their importance. We asked how stimulus type influences the effectiveness of dihydropyridine antagonists on CaV1.2 and CaV1.3 channel currents.

Nifedipine inhibits L-type currents

The dihydropyridine antagonist nifedipine is used widely to study neuronal L-type channels. We first confirmed that 5 μm nifedipine completely inhibited CaV1.2 currents activated by step depolarizations from a holding potential of -80 mV (Fig. 3a). Cells were exposed to maximum drug concentration within 1 s, and inhibition of CaV1.2 currents was complete within 12 s (six pulses) (Fig. 3a). In contrast, CaV1.3 channels were partially inhibited. Twenty percent of the CaV1.3 current remained in the presence of 5 μm nifedipine even after a 20 s drug exposure (Fig. 3b). These data show that 5 μm nifedipine completely inhibits CaV1.2 currents and, consistent with our previous studies in the Xenopus oocyte expression system (Lipscombe et al., 2004), nifedipine is less effective on neuronal CaV1.3 currents.

Slow inhibition of L-type currents opened by action potential waveforms

We next assessed the actions of nifedipine on neuronal L-type currents activated by action potential waveforms. The difference in the effectiveness of nifedipine on currents activated by these brief stimuli was striking. Nifedipine (5 μm) had no significant effect on CaV1.2 and CaV1.3 currents evoked by the first 30 action potentials of the stimulus train, applied from holding potentials of -80 and -60 mV (Fig. 4a-d). This same concentration of nifedipine strongly inhibited CaV1.2 and CaV1.3 currents activated by step depolarizations (Fig. 3a,b). Effects of nifedipine were only significant after 60 action potential stimuli (applied in consecutive trains of 30 action potentials from a holding potential of -80 mV; data not shown). Even after 90 pulses (three 30 pulse trains), a significant fraction of CaV1.2 and CaV1.3 currents remained unblocked at a holding potential of -80 mV (14.0 ± 4.8 and 38.1 ± 4.1%, respectively) (Fig. 4c,d). When activated from the more depolarized holding potential of -60 mV, CaV1.2 currents were inhibited completely after 90 pulses (Fig. 4c). Membrane depolarization promotes dihydropyridine inhibition (Bean, 1984), but even at the more depolarized membrane potential of -60 mV, significant inhibition was only observed after a series of stimuli. Our data are generally consistent with state-dependent inhibition; preferential inhibition of the inactive state of the L-type calcium channel by dihydropyridines promotes channel inactivation (Berjukow and Hering, 2001). CaV1.3 currents were, however, less sensitive to nifedipine, although CaV1.3 exhibited greater cumulative inactivation compared with CaV1.2 in control recordings (Fig. 4d, first two trains). Cumulative inactivation of CaV1.3 channels was calcium dependent (data not shown), and recovery from action potential-induced cumulative inactivation was rapid and complete in the absence of drug (Fig. 4d).

Nifedipine is weakly effective on CaV1.2 and CaV1.3 channels activated by action potential-like stimuli. Representative CaV1.2 (a) and CaV1.3 (b) currents evoked by a train of action potential waveforms, applied at 100 Hz from holding potentials of -80 mV, before (Con) and after a 10 s exposure to 5 μm nifedipine (Nif), are shown. Calibration: a, 0.2 nA, 50 ms; b, 0.5 nA, 50 ms. The action potential waveform used as command voltage was recorded from a sympathetic neuron and triggered by a brief current injection. Average peak current amplitudes for CaV1.2 (c; •, ○) and CaV1.3 (d; ▪, □) measured from currents induced by trains of 30 action potentials before and 10 s after exposure to 5 μm nifedipine (Nif) are shown. Currents were evoked from holding potentials of -80 mV (•, ▪) and -60 mV (○, □). Current amplitudes at the end of a series of four stimulus trains in the presence of nifedipine are shown for each series (180th pulse). Currents recovered completely after removal of nifedipine within three stimulus trains. Recovery was slowed approximately three fold when the membrane potential was depolarized to -60 mV. Error bars represent SE.

Discussion

We have studied the properties of the two most prevalent subtypes of neuronal L-type calcium channels in isolation from other currents. Our data show that both L-type channels open rapidly even in response to individual action potential waveforms, and CaV1.3 channels are as efficient in this regard as CaV2.2 N-type channels. Our findings are consistent with those by Liu et al. (2003), who found that a neuronal CaV1.2 channel clone responded to action potential stimuli with kinetics similar to CaV2.1 P/Q-type.

If neuronal L-type channels open rapidly, why has it been so difficult to establish their contribution to action potential-dependent calcium entry in regions abundant in these channels (Bonci et al., 1998; Mermelstein et al., 2000; Yasuda et al., 2003; Hoogland and Saggau, 2004)? Our study offers two possible explanations that both relate to the widespread use of dihydropyridine antagonists in studies of native neuronal L-type channels. First, in neurons that predominantly express CaV1.3, a significant fraction of L-type current will be resistant to dihydropyridine antagonists (Xu and Lipscombe, 2001). Second, dihydropyridine inhibition of CaV1.2 and CaV1.3 L-type channels develops slowly. In the short term, these drugs will have little effect on L-type currents activated by action potential stimuli triggered from resting membrane potentials; significant inhibition will develop with continued stimulation or if the membrane potential is depolarized for a prolonged period. Thus, ineffectiveness of dihydropyridines does not necessarily rule out L-type calcium channel involvement in neuronal processes.

It is important to note that channel properties, including inactivation, are influenced by several factors including the type of associating CaVβ subunit and the pattern of alternative splicing. These factors may also influence dihydropyridine effectiveness. In our experiments, we coexpressed CaVα2δ1 and CaVβ3 subunits with CaV1.2 and CaV1.3, but the specific isoform and subunit composition of each complex may vary with cell type and subcellular location (Birnbaumer et al., 1998; Lipscombe et al., 2002; Liu et al., 2003).

Conclusions

We suggest that the importance of L-type calcium channels to neuronal processes that are triggered by brief membrane depolarizations has been underestimated. L-type channels derived from neurons have intrinsic properties that suggest they can contribute to calcium-mediated processes triggered by a wide range of stimuli including gene expression, transmitter release, and rhythmic firing (Holz et al., 1988; Bonci et al., 1998; Lipscombe et al., 2004). Dihydropyridine antagonists are highly effective tools for establishing the involvement of L-type channels in processes triggered by prolonged periods of membrane depolarization, but of limited use for studying processes predominantly triggered by brief physiological stimuli, including action potentials, from rest.

Footnotes

This work was supported by National Institutes of Health Grants NS29967 (D.L.) and MH19118 (T.D.H.). We thank Alison Amenta for assisting in constructing CaV1.2 cDNA.